JPH0346540A - Detection of particulate material - Google Patents

Abstract

PURPOSE:To quantitatively determine the material to be detected regardless of the grain size of the product of agglutination reaction by adding a prescribed amt. of the particulate material of the specific diameter which induces the agglutination reaction with the material to be detected in a sample to the sample and irradiating the sample with the stimulating light adjusted to the wavelength equal to the diameter thereof. CONSTITUTION:The stimulating light 9 from a light source 1 is made incident to a cell 4. The prescribed amt. of the particulate material of the specific diameter which induces the agglutination reaction with the material to be detected is added to the sample in the cell 4 and the stimulating light 9 is absorbed by this material, by which an optoacoustic signal is generated. The sensitivity increases when the size of the particles coincides with the wavelength of the stimulating light 9 and, therefore, the particles of the coinciding diameter are added to the sample and are selectively detected and determined. The generated optoacoustic signal is detected by a detector of the cell 4 and the detected optoacoustic signal 12 is amplified by a lock-in amplifier 7 with the signal from an intensity modulator 2 as a reference signal 11. The intensity 14 of the measurement signal measured by such optoacoustic device is proportional to the concn. of the material to be detected. The calibration curve of the material to be detected is, therefore, obtd. by the intensity of the optoacoustic signal.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for detecting particulate matter in a liquid, and in particular,
The present invention relates to a photoacoustic analysis method suitable for immunoassay.

[Conventional technology]

In the conventional detection of particulate matter, as described in Japanese Patent Application Laid-Open No. 63-44149, excitation light was matched to the particle size of the agglutination reaction product in immunoassay. Therefore, consideration has not been given to the fact that the aggregation reaction product does not necessarily have a constant particle size.

[Problem to be solved by the invention]

The above conventional technology does not take into consideration the fact that the agglutination reaction product does not necessarily have a constant particle size, and does not necessarily detect the detection target substance in the sample that causes an agglutination reaction with added particles of a specific diameter with good sensitivity. There was a problem that could not be detected.

An object of the present invention is to provide a method for quantifying a substance to be detected in a sample regardless of the particle size of the agglutination reaction product.

[Means to solve the problem]

The above purpose is to add a predetermined amount of particulate matter with a specific diameter that causes an agglutination reaction with the detection target substance in the sample, and to apply excitation light whose wavelength is adjusted to be virtually equal to the diameter of the specific particulate matter to the sample. By irradiating, the amount of unreacted particulate matter with a specific diameter is detected, and the amount of added particulate matter with a specific diameter and the amount of unreacted particulate matter with a specific diameter are calculated. This can be achieved by detecting the concentration of the substance to be detected in the sample based on the difference in .

[Effect]

The present invention will be explained in detail below.

FIG. 1 shows an example of the basic configuration of the antigen-antibody unreacted particle quantification device according to the present invention. Excitation light 9 from a light source 1 such as a laser is incident on the cell 4. Particulate matter contained in the liquid sample within the cell 6 absorbs the excitation light and generates a photoacoustic signal. The generated photoacoustic signal is detected by a detector such as a piezoelectric element attached to the cell. The detected photoacoustic signal 12 is amplified by a lock-in amplifier 7 using a signal from an intensity modulator 2 such as an optical chopper as a reference signal 11. The intensity of the photoacoustic signal measured by such a photoacoustic device is proportional to the amount (concentration) of particulate matter contained in the sample. Therefore, a calibration curve of particulate matter based on the intensity of the photoacoustic signal can be obtained.

Next, FIG. 2 shows the particle size dependence of the sensitivity of the photoacoustic analyzer, that is, the slope of the calibration curve. The measurement results shown in Figure 2 were measured on polystyrene particles, and the excitation light intensity was 1.8 W, the wavelength was 488 nm, and the modulation frequency was 181 Hz.
This is a measurement by As shown in FIG. 2, when the particle size matches the excitation light wavelength of 488 nm (approximately 0.5 μm), the sensitivity of the photoacoustic spectrometer increases resonantly. Due to this effect, by adding particulate matter having a particle size that matches the wavelength of the excitation light 9 in FIG. 1 to a sample, it is possible to selectively detect and quantify the particles. In particular, if the particle size of the added particles changes due to aggregation due to an antigen-antibody reaction with the substance of interest in the sample, this photoacoustic spectrometer will no longer be able to detect it. Analytical target substances can be measured selectively and with high sensitivity.

〔Example〕

An embodiment of the present invention will be described below with reference to FIGS. 1 and 3.

The apparatus configuration of this embodiment is shown in FIG. The light source 1 is an Ar laser and oscillates a laser beam 9 of 488 nm. Laser light 9
is intensity-modulated into a rectangular wave with a modulation frequency of 181 Hz by a rotating blade type optical intensity modulator 2, that is, an optical chopper, and becomes excitation light 10.

Excitation light 10 enters cell 4 . The cell 4 is a cylindrical photoacoustic cell, in which a cylindrical piezoelectric element with the same inner diameter is built into the center of a glass cylinder, and this is used as a photoacoustic signal detector. The excitation light is incident along the central axis of the cylinder. A photoacoustic signal generated when the cell 4 is filled with a liquid sample and the excitation light 10 is incident is converted into an electrical signal by a piezoelectric element.

The photoacoustic signal 12 converted into an electrical signal is input to the lock-in amplifier 7. The lock-in amplifier 7 extracts only the photoacoustic signal corresponding to the excitation light 10 from the detected photoacoustic signal 12 using the signal 11 synchronized with the modulation of the optical intensity detector 2 as a reference signal 4-, and extracts the photoacoustic signal corresponding to the excitation light 10 from the detected photoacoustic signal 12. Input intensity 14 into recorder 8.

Examples in which the present invention is applied to immunoassay will be described below. α-Fetoprotein (hereinafter abbreviated as AFP) is measured using the above-mentioned apparatus.

(1) Preparation of sample for AFP measurement Anti-human AFP antibody in 0.1M glycine buffer (pH 6)
, 5) Add 1 mQ of white polystyrene latex (solid content concentration 10%) with a particle size of 0.5 μm to 10 m + 2, stir at room temperature for 3 hours, and then cool for 40 minutes at 2 to 4 ° C.
Centrifugation was performed at 1200 rpm. The obtained precipitate was reduced to 0
．． 5% bovine serum albumin, 0.1M NaCQ, 2mM
10mM phosphate buffer containing gCf1z (pH 6,5)
Anti-AF with a concentration of 1% sensitized latex particles
A P antibody sensitized latex reagent was prepared.

(2) Measurement of sample Sample solution: 10 μl of bovine serum albumin solution added with human AFP. 50 μl of anti-human AFP antibody sensitized latex reagent prepared with (1).

A 200μ flood of 10mM phosphate buffer solution (pH 6.5) was added and anti-antigen reaction was carried out at 37°C for 10 minutes. This reaction solution was filled into a photoacoustic cell, and the photoacoustic signal was measured.

〔Effect of the invention〕

According to the present invention, the detection target substance can be quantified regardless of the particle size of the aggregation reaction product of the detection target substance in the sample and the added particulate additive substance having a specific diameter, so that the following effects can be achieved. That is, (1), even if the particle size of the aggregation reaction product is not constant,
The substance to be detected can be accurately quantified.

(2) By matching the diameter of the particulate matter added to the sample with the excitation light, a wavelength sweeping mechanism for the excitation light becomes unnecessary.

[Brief explanation of drawings]

Fig. 1 is a block diagram of an embodiment of the present invention, Fig. 2 is a characteristic diagram showing the dependence of sensitivity on particle size, and Fig. 3 is a characteristic diagram showing the calibration curve of alpha-fetoprotein using the apparatus shown in Fig. 1. It is a diagram. 1. Light source, 2. Intensity modulator, 3. Half mirror 4.
・Cell, 5 ・Beam stopper, 6...Light intensity monitor,
7.Lock-in up, 8.Recorder, 9.Laser light, 10.Excitation light, ]1..Reference signal, 12..
Photoacoustic signal, ↓3 Monitor signal, 14...Signal strength.

Claims (1)

[Claims] 1. A method for determining the concentration of particulate matter having a diameter equal to the wavelength of the excitation light by irradiating particulate matter in a sample with excitation light, the method comprising: exposing part of the sample to the sample; By adding a predetermined amount of particulate matter with a specific diameter that causes an agglutination reaction with the components of Detecting the amount of reacting particulate matter with a specific diameter, and determining the difference between the amount of added particulate matter with the specific diameter and the amount of unreacted particulate matter with the specific diameter. , a photoacoustic analysis method for detecting the amount of particulate matter having a specific diameter that has aggregated with some components of the sample. 2. The photoacoustic analysis method according to claim 1, wherein the concentration of some components of the sample is determined from the amount of the aggregated particulate matter having a specific diameter.